Phenolic Acid Composition and Anti-Parasitic Effects of Four Peucedanum Species on Entamoeba histolytica Trophozoites.

BACKGROUND
The aim of this study was to determine phenolic acid composition and anti-parasitic effects of Peucedanum caucasicum, P. palimbioides, P. longibracteolatum and P. chryseum on Entamoeba histolytica.


METHODS
Methanol extracts of the plant species were prepared by soxhlet extraction. Phenolic acid compositions were determined by HPLC. Anti-proliferative effect of extracts on trophozoites was determined by using trypan blue dye exclusion test. For counting the cells, approximately a hundred of E. histolytica trophozoites were examined in each time. The data were presented as mean values with standard deviations and analyzed by repeated measures of ANOVA followed by Tukey test for post-hoc pairwise comparisons. The P-value was set at 0.05 for significance level.


RESULTS
All of the extracts showed a time and dose dependent amoebicidal action on trophozoites. Among the extracts tested, P. longibracteolatum showed the strongest amoebicidal effect on the trophozoites. As expected, this plant species also exhibited time and dose dependent activity on the trophozoites. At 4.0 mg/ml extract concentration, all of the trophozoites were killed by the extract in 72(nd) hour. Gallic (11.144 mg/g), P-hydroxybenzoic (17.646 mg/g), and o-coumaric acids (14.442 mg/g) were determined as the major phenolic acids of P. longibracteolatum. Gallic and P-hydroxybenzoic acids found in P. longibracteolatum could not be determined in other extracts. Therefore, high activity potential of this plant could probably be attributed to the presence of these phytochemicals.


CONCLUSION
P. longibracteolatum can be further evaluated as potential therapeutic drugs for the treatment of Entamoeba infections.

Introduction eath from protozoan parasite, Entamoeba histolytica, is still one of the important health problems in developing countries that also known as third world countries. Among these deaths, amoebiasis takes the second place (1). It affects more than 10% of the world's population. Untreated infections may cause to severe complications such as hepatic amoebiasis and intestinal tissue destruction (2). Amoebiasis is responsible for approximately 110,000 deaths worldwide annually. The main reason for about 50 million clinical cases each year is estimated as amoebiasis (3,4).
E. histolytica/dispar is responsible for an estimated 35 to 50 million cases of disease and approximately 100 000 deaths annually (5). In general, most of morbidity and mortality are seen in Asia, Central and South America and Africa.
The most prevalence of amoebiasis was seen in 10-35 yr age groups globally. Children suffer from malnourishment and stunting as the result of infections occurs repeatedly (6). Infection resistance is directly related with the production of interferon-g and mucosal IgA produced by peripheral blood mononuclear cells. These agents are directed to the parasite surface lectin (7). Studies are underway to develop an effective vaccine. For treatment of invasive amoebiasis, limited numbers of agents mainly nitroimidazoles such as metronidazole are available today. However, toxic side effects can occur because of the treatment of infection with these drugs. Additionally, there is a need for additional drugs in 40-60% of patients to cure infection (8).
The prevalence of infection is known as due to the location and climate of the region where people live. However, it is closely related to sanitation and socioeconomic status of people rather than these factors. Poor lifestyle, environmental conditions of the tropics, and the non-availability of guaranteed conven-tional medical cure are among the complex etiological factors (9).
The genus Peucedanum L. (Apiaceae) is mainly distributed in Europe and Asia. It comprises about 100-120 species (10). Peucedanum species in Turkish flora are represented by 18 taxa (with 19 species, 2 of which are suspected). Among these species, seven are endemic to Turkey with an endemism rate of 28.5% (11)(12)(13)(14)(15)(16). Peucedanum species are known as "kral out" in Turkey and they are under investigation due to their substantial amounts of chemically active phytochemicals, such as coumarins and their glycoside derivatives (10,17,18). Some members of this genus (P. dissolutum and P. praeruptorum) are used in traditional Chinese medicine (Qianhu) to cure ailments such as coughs, phlegm, and other respiratory illnesses (18,19). Additionally, essential oil compositions, antimicrobial and antiproliferative activities of the some members of this genus have been investigated (18)(19)(20).
The aim of this study was to determine in vitro susceptibility of P. caucasicum, P. palimbioides, P. longibracteolatum and P. chryseum for the treatment of gastrointestinal disorders. Additionally, determination of the phenolic acid composition of the samples was also aimed to have an idea about the relative contribution of these phytochemicals on anti-parasitic action. According to our literature survey, antiparasitic activities of these plants have not previously been reported. Therefore, this study could be assumed as the first report on this topic.

Preparation of the methanol extracts
The air-dried and finely ground samples were extracted by using a method described elsewhere (36). Briefly, the sample, weighing about 100 g, was extracted in a Soxhlet apparatus with methanol (MeOH) at 60 ºC for 6 h. Due to the aqueous characteristics of the experimental media; mainly the water-soluble parts of the extracts could be used only. For obtaining the aqueous sub-fraction of the extracts, it was further fractionated with chloroform and distilled water. Finally, the extracts were then lyophilized and kept in the dark at +4 ºC until tested. Extract yields of the polar sub-fractions of the rhizome and aerial part samples were determined as 2.24%, 3.12%, 3.56%, and 3.40% w/w, respectively.

Trophozoites
E. histolytica strain was kindly provided by Dr. Graham Clark who is working in London School of Hygiene & Tropical Medicine and he is a specialist on amoebiasis. It was a pathogenic strain isolated from a Bangladeshi child with acute amoebic dysentery. Trophozoite forms of parasite were cultured monoxenically in the Robinson medium at 35.5 C° (37). Agar slants were prepared in screw-cap glass bijou bottles, 1.5 ml of phthalate, 1.5 ml of BRS medium, 50 μl of erythromycin and 100 μl Bacto peptone were mixed in the bottle and sterilized rice starch and Escherichia coli R medium are added to the medium before inoculation. Each time 0.5 ml of culture is used for inoculation and culture was renewed for a further 96 h to maintain the amoeba strain. The trophozoites in the log phase of growth were harvested by centrifugation at 500g at room temperature for 5 min. The pelleted cells were resuspended in Phosphate Buffer Saline (PBS), the final concentration was adjusted to 10x10 4 trophozoites/ml and the trophozoites were used in the assay immediately. The number of viable trophozoites was determined by trypan blue exclusion and direct trophozoite counts on a hemocytometer prior to experiment.

Determination the amoebicidal activity of plant extracts
Eppendorf tubes were used in the present study. The plant extracts were dissolved and diluted in sterile distilled water to serial concentrations (2,4,8,16,32 and 64 mg/ml). Totally, 250 µl of the calibrated trophozoite suspension and the same volume of test solution were mixed thoroughly via pipetting up and down. Then the tubes were kept at 35.5 °C in standard bacteriological incubator (Electro-Mag) for 1, 2, 3, 6, 8, 24, 48 and 72 hours. The same procedure was applied to controls containing only trophozoite suspension and sterile distilled water. Cell growth was periodically monitored by light microscopy (Nikon, Eclipse E 200).
Trypan Blue Dye Exclusion Test was used to determine the effect of extracts on the proliferation of parasite. Following the incubation periods at 35.5 °C, 25 μl parasite suspensions was simply mixed with the same volume of trypan blue (0.5 mg/ml) in counting chamber. The mixture was allowed to incubate 2-3 min at room temperature and then the unstained (viable) and stained (nonviable) cells were counted separately. Approximately a hundred of E. histolytica trophozoites were examined in each time, and all the tests were repeated three times.

Statistical analysis
The data were presented as mean values with standard deviations and analyzed by repeated measures of ANOVA followed by Tukey test for post-hoc pairwise comparisons. The Pvalue was set at 0.05 for significance level.

Results
In vitro amoebicidal activity of the polar extract of P. caucasicum extract on E. histolytica trophozoites is presented in Table 1. O. syriacum extract showed the weakest trophozoidal activity in this study. At 32.0 mg/ml concentration, within 72 nd hour, approximately, 60% of the trophozoites were killed by the extract.
Susceptibility of E. histolytica trophozoites to the polar extract of P. palimbioides is also presented in Table 2. P. palimbioides extract showed remarkable trophozoidal activity. At 16.0 mg/ml concentration, the extract killed 50% of the trophozoites in 8 th hour. At this concentration, within the 72 nd hour, approximately 80% of the trophozoites were killed. At 32.0 mg/ml concentration, number of the viable trophozoites was 10% within 72 nd hour.
One another plant species of which amoebicidal activity investigated was P. longibracteolatum (Fig. 1). Polar methanolic extract obtained from this plant species was applied to E. histolytica trophozoites (Table 3). This extract showed the strongest activity potential on the trophozoites. As expected, this plant species also exhibited time and dose dependent activity on the trophozoites. At 4.0 mg/ml extract concentration, all of the trophozoites were killed by the extract in 72 nd hour. The extract at 8.0 mg/ml concentration was also killed all of the trophozoites in 48 th hour. In the case of 16.0 and 32.0 concentrations, after 8 th hour, no viable trophozoites were observed in the media.
Finally, in vitro amoebicidal activity of the polar methanol extract of P. chryseum was investigated on E. histolytica trophozoites ( Table  4). The extract at 32.0 mg/ml, exhibited great activity potential. At 32.0 mg/ml concentration, more than 50% of the trophozoites were killed effectively in 24 th hour. At the end of the experimental process, the number of the viable trophozoites was determined as 15.3% at the same concentration level.

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Available at: http://ijpa.tums.ac.ir Each row was evaluated individually from the statistical point of view.   For making a better comparison between the results presented in Tables 1-4, data obtained from the 32.0 mg/ml concentration was also presented in Fig. 2.
By this study, we also reported the strongest amoebicidal effect of P. longibracteolatum on the trophozoites and cysts. From the twentyfourth hour, no viable trophozoites or cysts were determined at 32 mg/ml of extract concentration. Similar results were also obtained with the extract at 16.0 mg/ml concentration. At this concentration, number of viable cysts was determined as 10.6 from the twenty-fourth hour of experimental process. At 8.0 mg/ml extract concentration, 51% of the cysts were killed by the extract in 72 nd hour. Apart from the anti-parasitic activities, plant species evaluated here are also screened for their phenolic acid compositions. By this way, we tried to have an idea about the relative contribution of these phytochemicals in antiparasitic potential of extracts. For this purpose, twelve different phenolic acids including; gallic acid, protocatechuic acid, P-hydroxybenzoic acid, vanillic acid, caffeic acid, chlorogenic acid, syringic acid, P-coumaric acid, ferulic acid, rosmarinic acid, o-coumaric acid, transcinnamic acid were screened within the extracts. Peaks of standard phenolic acids used are given as a chromatogram in Fig. 3. HPLC chromatograms of the plant species showing phenolic acid compositions are presented as Fig. 4-7.
Vanillic, chlorogenic, rosmarinic, gallic, Phydroxybenzoic, and o-coumaric acids were determined within the extracts. The most common phenolic acid was determined as vanillic acid that found in P. caucasicum, P. palimbioides, and P. chryseum. As seen from Table 3, P. longibracteolatum showed the highest antiparasitic activity against E. histolytica trophozoites. According to HPLC analysis of this sample, gallic (11.144 mg/g), P-hydroxybenzoic (17.646 mg/g), and o-coumaric acids (14.442 mg/g) were determined as the major phenolic acids (Fig. 6). Plant containing the maximum number of phenolic acids was P. chryseum of which main compounds were determined as gallic (1.347 mg/g), vanillic (3.014 mg/g), o-coumaric (8.420 mg/g), and rosmarinic acids (4.642 mg/g) (Fig. 7). As can be seen from Table 2, anti-parasitic activity of P. longibracteolatum was followed by P. palimbioides. According to chromatographic analyses, a similar phenolic acid profile was observed for this plant (Fig. 5). The main phenolic acids of this plant were determined as vanillic (14.640 mg/g), chlorogenic (29.215 mg/g), and rosmarinic acids (6.577 mg/g). Phenolic composition of P. caucasicum was also determined chromatograph-ically (Fig. 4). Vanillic (7.167 mg/g) and chlorogenic acids (4.885 mg/g) were found as the major phenolic acids among the standards screened.

Discussion
Enteric protozoan infections are among the important causes of morbidity and mortality. They are still known as the serious health problems in developing countries. Abdominal pain and tenderness, diarrhea, and bloody stools are among the most common symptoms frequently observed in patients having an Entamoeba infection. By the treatment amoebiasis, eliminating clinical symptoms and destroying amoeba in its cyst or trophozoite forms are aimed. Amoebicides are frequently administered to the patients orally, intramuscularly and intravenously. Currently, in the treatment of amoebic infections such as dysentery and liver abscesses, just a few amoebicides can be used due to their narrow therapeutic uses and undesirable side effects on patients. In dysentery, metronidazole is the main drug of choice. It is known as a highly effective agent against Entamoeba. However, this drug has mutagenic effects in bacteria and is carcinogenic in some animals. On the other hand, headache, dry mouth, a metallic taste, and neurotoxicity and gastrointestinal disturbances, especially nausea is among the most common side effects of metronidazole. In some patients, vomiting and diarrhoea or constipation may also occur (38,39). E. histolytica can show multidrug resistance during the treatment (40). Metronidazole resistance was induced experimentally in the E. histolytica axenic strain HTH-56: MUTM (41).
Plant extracts possess a wide array of biological activities that can be easily used for the betterment of human beings. Many plants, used in traditional medicine, proved to be of immense importance and gave significant results in having antiamoebic activities. A detailed study of plants with antiamoebic properties should be done based on the ethnobotanical leads available. However, these findings can only be of practical use when they would undergo stringent clinical trials (42).
Among the extracts tested, P. longibracteolatum showed the strongest amoebicidal effect on the trophozoites (Fig. 1).. As expected, this plant species also exhibited time and dose dependent activity on the trophozoites. At 4.0 mg/ml extract concentration, all of the trophozoites were killed by the extract in 72 nd hour. The extract at 8.0 mg/ml concentration was also killed all of the trophozoites in 48 th hour.
Materials extracted with methanol were further fractionated with chloroform and water to obtain polar and non-polar phytochemicals separately. In experimental process presented here, it is necessary using the polar components to determine their amoebicidal activities due to the polarity of media that requires the using polar substances. Polar compounds can enter chemical reactions easier than those of non-polar ones within the cells can. By this way, they can show desired activity potential more effectively.
In general, we could not establish a direct relationship between phenolic acid composition and anti-parasitic activity potential of plants. However, as can be seen from the chromatograms, gallic and P-hydroxybenzoic acids mainly found in P. longibracteolatum extract could not be determined in other extracts. Therefore, high activity potential of this plant could probably be attributed to the presence of these phytochemicals. However, in order to have a clearer judgment on this topic, antiparasitic activities of individual phenolic acid components should also be evaluated.

Conclusion
P. longibracteolatum showed strongest activity potential on the proliferation of E. histolytica trophozoites. This activity was followed by P. palimbioides, P. chryseum, and P. caucasicum, respectively. These results suggest that P. longi-bracteolatum can be further evaluated as potential therapeutic drugs for the treatment of Entamoeba infections.